Programmable activation of Bombyx gene expression using CRISPR/dCas9 fusion systems

Area of Science:

• Molecular Biology
• Genetics
• Insect Science

Background:

• CRISPR-based gene editing techniques enable targeted gene expression reprogramming.
• Previous studies demonstrated CRISPR-Cas9 and CRISPR-Cpf1 for gene modification in Bombyx mori.
• The applicability of the CRISPR activation (CRISPRa) system in B. mori remained unexplored.

Purpose of the Study:

• To investigate the efficacy of the CRISPRa system for gene upregulation in Bombyx mori.
• To compare the performance of dCas9-VP64 and dCas9-VPR systems in B. mori cells.
• To assess the potential of CRISPRa as a tool for functional genomics in insects.

Main Methods:

• Utilized a nuclease-dead Streptococcus pyogenes Cas9 (SpCas9) fused to transcription activation domains (VP64 and VPR).
• Introduced dCas9-VP64 and dCas9-VPR systems into B. mori cells to activate target genes.
• Analyzed gene expression levels and correlated upregulation with basal expression.

Main Results:

• Both dCas9-VP64 and dCas9-VPR systems were functional in B. mori cells.
• The dCas9-VPR system exhibited significantly higher gene activation efficiency.
• Significant upregulation was observed across five tested target genes, inversely correlated with their basal expression levels.

Conclusions:

• The CRISPRa system, particularly dCas9-VPR, is a potent tool for gene function studies in Bombyx mori.
• This technology offers a new avenue for functional genomics research in non-drosophila insects.
• CRISPRa facilitates targeted gene upregulation without genomic alteration or cDNA cloning.